Gasdermin D Deficiency in Vascular Smooth Muscle Cells Ameliorates Abdominal Aortic Aneurysm Through Reducing Putrescine Synthesis

Abstract Abdominal aortic aneurysm (AAA) is a common vascular disease associated with significant phenotypic alterations in vascular smooth muscle cells (VSMCs). Gasdermin D (GSDMD) is a pore‐forming effector of pyroptosis. In this study, the role of VSMC‐specific GSDMD in the phenotypic alteration of VSMCs and AAA formation is determined. Single‐cell transcriptome analyses reveal Gsdmd upregulation in aortic VSMCs in angiotensin (Ang) II‐induced AAA. VSMC‐specific Gsdmd deletion ameliorates Ang II‐induced AAA in apolipoprotein E (ApoE)−/− mice. Using untargeted metabolomic analysis, it is found that putrescine is significantly reduced in the plasma and aortic tissues of VSMC‐specific GSDMD deficient mice. High putrescine levels trigger a pro‐inflammatory phenotype in VSMCs and increase susceptibility to Ang II‐induced AAA formation in mice. In a population‐based study, a high level of putrescine in plasma is associated with the risk of AAA (p < 2.2 × 10−16), consistent with the animal data. Mechanistically, GSDMD enhances endoplasmic reticulum stress‐C/EBP homologous protein (CHOP) signaling, which in turn promotes the expression of ornithine decarboxylase 1 (ODC1), the enzyme responsible for increased putrescine levels. Treatment with the ODC1 inhibitor, difluoromethylornithine, reduces AAA formation in Ang II‐infused ApoE−/− mice. The findings suggest that putrescine is a potential biomarker and target for AAA treatment.

3 aortas were harvested, fixed, and cleaned for maximal outer diameter measurements in the infrarenal region. Image J was used to measure the maximum outer diameter of aortas.

Mass Spectrometry
For plasma samples, fasting blood samples were collected into K2 EDTA vacutainer tubes and immediately placed in a refrigerator (4 °C). Samples were centrifuged within 2 h at 3000 g for 10 min at 4 °C. Supernatants (plasma) were separated and transferred into new vials, and frozen (-80 °C) until preparation. Frozen plasma samples were thawed at 4 °C. A total of 100 μL of plasma was spiked with 5 μL of putrescine (13C4) (5 μM), followed by addition of 300 μL of methanol. After vortex mixing for 4 min and centrifuging at 14,000 rpm for 4 min at 4°C, the supernatant was separated, then add a certain volume of acidic water to the supernatant, mix well and wait for loading the sample.
For tissue samples, frozen tissue with the same wet weight was accurately weighed into a 2 mL homogenization tube containing four ceramic beads (3.0 mm diameter). Pre-cooled extraction solvents (500uL of 80%(v/v) HPLC-grade methanol, which contains 5 μM of putrescine (13C4), were added, and the tissue was homogenized three times for 30 s at a shock velocity of 4.0 m/s using a high-throughput tissue homogenizer. After homogenization, samples were centrifuged at 14,000 rpm for 4 min at 4°C. Supernatant were separated, then acidic water was addedto the supernatant and mixed well.
All the above mixed supernatant was then passed through solid-phase extraction (SPE) cartridges (Oasis MCX 3cc cartridges, 60 mg, Waters, Milford, MA, USA). The sorbent was conditioned with 3 mL methanol and equilibrated with 3 mL of water before samples were 4 applied. After supernatants were eluted, 2 mL water and 2 mL methanol were passed through cartridges, and then eluted with 2 ml ammoniated methanol, the eluate was collected and dried using a Savant concentrator with RVT-5105 refrigerated vapor. Before analysis, the extracts were resuspended in 100 ul of 0.5% FA/water (v/v). A 5 μL aliquot of sample was injected into the LC-MS system for analysis.
The chromatography was performed on a Shimadzu Prominence system with a binary pump, an online degasser, an autosampler and a column oven (Shimadzu Scientific Instruments, INC., Columbia, MD, USA). The separation was achieved on a Waters ACQUITY HSS T3 column Declustering potentials and collision energy were 28 V and 13 eV for putrescine, 15 V and 11 eV for IS, respectively. Collision exit potential were 9 V for all analytes. The mass spectrometer was operated under the following optimum parameters: curtain gas (nitrogen), 30; ionspray voltage, 5500 V; source temperature, 550°C; ion source gas 1 (zero-grade air), 55; ion source gas 2 (zero-grade air), 60; and collision gas (nitrogen), LOW. Analyst software 1.7.3 (Sciex) 5 was used for the data acquisition as well as processing.

Histology
Aortas were fixed in 4% paraformaldehyde overnight. After dehydration with 20% sucrose, aortas were embedded in optimal cutting temperature compound and sliced with a cryostat with 7μm tissue sections. Sections were stained with Elastin Van Gieson (EVG) and hematoxylineosin (HE). Sections were imaged using a BX43 Olympus microscope (Japan). Representative histological photomicrographs are presented. For the grade of elastin degradation, the definition is as followed: grade 1, no degradation; grade 2, mild elastin degradation; grade 3, severe elastin degradation; and grade 4, aortic rupture.

Immunohistochemistry
Frozen sections were removed from a freezer and placed at room temperature for 30 minutes.
The sections were immunostained using a rabbit anti-mouse GSDMD antibody (1:200 dilution, Cat#ab219800, abcam, UK). Secondary antibody used were biotinylated goat anti-rabbit IgG (Cat#PV9000, ZSGB-BIO, China). Negative controls included non-immune rabbit IgG2a, no primary antibody control, and no primary and secondary antibody control. Positive immunoreactivity was visualized as red color by oxidation of aminoethyl carbazole. Sections were imaged using a BX43 Olympus microscope.

Immunofluorescence
Frozen sections were removed from a freezer and placed at room temperature for 30 minutes. 6 Sections were incubated with primary antibodies at 4°C overnight, and then incubated with secondary antibodies for 60 min at room temperature. Isotype match non-immune IgG2a, omission of primary antibody, and omission of both primary and secondary antibodies were used as negative controls. Mounting medium with DAPI (ZSGB-BIO, ZLI-9557, China) was applied for nuclear staining. Sections were imaged through a Leica TCS SP8 STED confocal fluorescent microscope (3X, Germany). Smooth muscle cells were fixed with 2.5% glutaraldehyde for 1h at room temperature. Samples were fixed with 1% osmium tetroxide for 1h, dehydrated through ethanol, and embedded in Epon. The ultramicrotome (Leica EM UC6 C) was used to make ultrathin sections (70 nm).
The sections mounted on copper grids were stained with uranyl acetate and lead citrate and then examined by the transmission electron microscopy system (Talos L120C).
Senescence-associated-β-galactosidase (SA-β-gal) staining By using the kit of SA-β-gal (C0602, Beyotime Biotechnology, China), cultured cells were washed in ice-cold PBS for three times and then fixed for 10 min at room temperature. After washing for three times, the cells were incubated in staining buffer for 12-hours at 37℃. After the stain, the cell could keep in ice-cold PBS for a few days. SA-β-gal images were analyzed with Image J software. The average optical density of the senescence (blue) was evaluated.